Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA, USA.

Abstract

Post-traumatic stress disorder (PTSD) and alcoholism are highly comorbid in humans and have partially overlapping symptomatic profiles. The aim of these studies was to examine the effects of traumatic stress (and stress reactivity) on alcohol-related behaviors and neuronal activation patterns. Male Wistar rats were trained to respond for alcohol, were exposed to predator odor (bobcat urine) paired with context and were tested for short- and long-term avoidance of the predator odor-paired context, alcohol self-administration and compulsivity of alcohol responding. Rats were re-exposed to the odor-paired context for western blot analysis of ERK phosphorylation in subregions of the medial prefrontal cortex (mPFC) and the amygdala. Rats that avoided the predator-paired chamber (Avoiders) exhibited persistent avoidance up to 6 weeks post conditioning. Avoiders exhibited increases in operant alcohol responding over weeks, as well as more compulsive-like responding for alcohol adulterated with quinine. Following re-exposure to the predator odor-paired context, Avoiders and Non-Avoiders exhibited unique patterns of neuronal activation in subregions of the mPFC and the amygdala, which were correlated with changes in avoidance and alcohol drinking. Furthermore, activity of upstream regions was differentially predictive of downstream regional activity in the Avoiders versus Non-Avoiders. An animal model for assessing the effect of traumatic stress on alcohol drinking reveals individual differences in neuronal activation patterns associated with re-exposure to traumatic stress-related stimuli, and may provide insight into the neural mechanisms underlying excessive alcohol consumption in humans with PTSD.

(a) Mean (±s.e.m.) change in time (s) spent in predator-paired context from pre-conditioning test to post-conditioning test, quantified as post-test score minus pre-test score. Rats (Experiment 1) were divided into Avoiders (n=23) and Non-Avoiders (n=14) based on avoidance of predator-paired chamber at 24 h post conditioning, and then were tested again for avoidance 6 weeks later. Rats that exhibited high avoidance of the predator-paired chamber at 24 h post-conditioning also exhibited avoidance 6 weeks later. (b) Scatter plot for individual rat (Experiment 3) shows change in preference for a context repeatedly paired with an appetitive stimulus (saccharin+glucose solution) versus change in preference for a context repeatedly paired with predator odor. There was no predictive value by the degree to which a rat conditioned the appetitive stimulus for the degree to which a rat conditioned the aversive stimulus. This also suggests that differences in avoidance are not attributable to differences in ability of rats to learn. (c) Scatter plot for individual rat (Experiment 3) shows percent time spent in open arms of EPM (5-min test) in experimentally naive rats (index of innate anxiety-like behavior) versus change in preference for a context repeatedly paired with predator odor. There was no predictive value by innate anxiety-like behavior for the degree to which a rat conditioned the aversive stimulus. (d) Mean operant alcohol responses (±s.e.m.) per 30 min session for Avoider, Non-Avoider and non-stressed control groups (Experiments 1 and 2 combined) across the last 3 days of the pre-conditioning baseline self-administration period. Groups did not differ in baseline alcohol self-administration. These data also provide further support for the notion that differences in avoidance are not attributable to differences in ability of rats to learn. (e) Mean (±s.e.m.) operant presses for alcohol per 30-min self-administration session by rats in Experiment 2 (results similar in Experiment 1, see text) during intermittent testing across the 19 days following exposure to predator odor (Avoider and Non-Avoider groups) or no odor (control group). Avoider rats exhibited persistent increases in operant alcohol responding relative to the two other groups and their own baseline across the 19 days following exposure to odor. *P<0.05 significant main effect of group. Avoiders exhibited marginally non-significant increases in alcohol responding relative to Non-Avoiders (P=0.06) and unstressed Controls (P=0.055). (f) Mean (±s.e.m.) self-administration of alcohol solution adulterated with progressively increasing quantities of quinine, quantified as percent change from baseline (10% w/v ethanol plus 0% quinine) for each rat (Experiment 1). A 10 × higher concentration (0.025% vs 0.0025%) of quinine was required to reduce operant alcohol self-administration in Avoider rats vs Non-Avoider and Control rats, indicative of more compulsive-like alcohol drinking in Avoider rats. **P<0.001 Avoiders responded significantly more for alcohol + 0.0025% quinine relative to unstressed Controls. There was a non-significant tendency of Avoiders to press more for this solution than Non-Avoiders (P=0.10). (g) Scatter plot for individual rat (Experiment 2) shows change in preference for predator-paired context versus percent change in operant alcohol responding at day 19 post-odor exposure relative to baseline. Rats that exhibited high avoidance of the predator-paired context 24 h post-odor exposure self-administered more alcohol at 19 days post-odor exposure.

(a) ERK phosphorylation (pERK) in the medial prefrontal cortex (PFC) after re-exposure (15 min) to the predator odor-paired context. Relative to neutral-paired animals (controls), re-exposure to the context produced a bidirectional regulation of pERK levels in the vmPFC in the odor-paired groups, with Avoiders displaying relatively higher ERK phosphorylation compared with that of Non-Avoiders. This relationship was not observed in the dorsomedial PFC (dmPFC). **P<0.01 higher ERK phosphorylation levels in Avoiders versus Non-Avoiders or *P<0.05 higher ERK phosphorylation levels in vmPFC versus dmPFC in Avoiders. (b) ERK phosphorylation in the amygdala subregions after re-exposure to the odor-paired context. As a percentage of neutral-paired control animals, Avoider and Non-Avoider stress-paired groups displayed more ERK phosphorylation in the central nucleus of the amygdala (CeA) versus basolateral amygdala (BLA). **P<0.01 higher ERK phosphorylation levels in the CeA versus BLA of pooled stress-paired groups by analysis of variance (main effect of group). Data reflect the mean±s.e.m. of pERK/ERK ratios and are expressed as a percentage of neutral-paired controls. Inset: representative western blot images for pERK and ERK levels in vmPFC, CeA and BLA in Control (C), Avoider (A) and Non-Avoider (NA) groups.

Within-subject between-region correlations of ERK phosphorylation upon re-exposure to the predator odor-paired context. (a) In Avoiders (left panel), but not in Non-Avoiders (right panel) or Controls (data not shown), there was a significant correlation between dorsomedial prefrontal cortex (dmPFC) and basolateral amygdala (BLA) ERK phosphorylation (pERK) expression. Panel b reports the strength and direction of correlations between pERK expression in ventromedial PFC (vmPFC) and central nucleus of the amygdala (CeA; top row), and also between BLA and CeA (bottom row) in Avoiders, Non-Avoiders and Controls upon re-exposure to predator odor-paired context.

Relationship between alterations in individual levels of alcohol drinking (19 days post conditioning) and odor-paired-context-induced ERK phosphorylation in the dorsomedial prefrontal cortex (dmPFC) and basolateral amygdala (BLA). Elevations in drinking after odor exposure significantly correlated with context-induced reductions in ERK phosphorylation (pERK) levels in both the dmPFC (a, r2=0.25, P<0.05) and BLA (b, r2=0.24, P<0.05) of Avoiders, but not of Non-Avoiders or Controls.

Proposed model of corticolimbic neurocircuitry underlying traumatic stress-induced avoidance and increases in excessive alcohol drinking. Traumatic stress reminder cue-induced neuronal activation (that is, ERK phosphorylation (pERK) expression) in the ventromedial prefrontal cortex (vmPFC) predicts high avoidance behavior. Also depicted here is that the hyporeactivity of the dorsomedial PFC (dmPFC) and basolateral amygdala (BLA) to a traumatic stress reminder is highly predictive of each other and is also highly predictive of escalated post-stress alcohol drinking only in Avoider rats, suggesting an increased dmPFC–BLA connectivity in Avoider rats. Two potentially important aspects of this circuitry are not depicted here: (1) the population of intercalated γ-aminobutyric acid (GABA) cells that act as a relay for some but not all central nucleus of the amygdala (CeA) afferents, and (2) the neuroendocrine (hypothalamic–pituitary–adrenal) stress axis that is driven by vmPFC activity and inhibited by dmPFC activity,, and which itself may interact with the CeA in a reciprocally excitatory loop.